PROJECT SUMMARY/ABSTRACT Doxorubicin (DOX) is a highly effective chemotherapy agent that is commonly used in combination with precision medicine to treat a wide range of cancers, including 32% of breast cancer (BC) cases. Although the treatment has greatly increased the number of long-term cancer survivors, it has also increased the number of patients experiencing DOX-induced cardiotoxicity (DIC). Currently, there are no validated biomarkers that can predict the early development of DIC. Cardiac troponin released by cardiomyocytes has been used in non-clinical studies as a marker of myocardial injury; however, it has a low specificity, and the predictive value on cardiotoxicity is limited. Therefore, novel biomarkers of DIC are urgently needed to identify patients who are at an increased risk, allowing early detection of the cardiotoxicity before it causes permanent cardiac damage. Recent studies have identified new pathways that are altered in the heart during DIC, providing opportunities for the identification of early biomarkers of cardiac toxicity. DOX is reduced by mitochondrial complex I to form a semiquinone free radical, resulting in the oxidation of a variety of cellular molecules, including lipid peroxidation and oxidized phospholipids. These metabolic alterations can be leveraged to develop DIC biomarkers. Metabolomics identifies perturbations in molecular pathways due to toxicity an?d disease and is ideally suited for identifying early indications of DIC. Previously, our research team reported early metabolite changes in the heart and plasma of DOX-treated mice and identified altered levels of Krebs cycle related metabolites at the lowest cumulative dose of DOX, before troponins are released into the blood and cardiac lesions appear. Our goal is to identify early metabolite markers of cardiotoxicity in blood that are associated with cardiac dysfunction. In collaboration with the PI's 2 COBRE mentors, our lab is part of an ongoing clinical study of DOX-treated BC patients. We have previously identified cohorts of patients who developed an abnormal decline in left ventricular ejection fraction and patients who maintained normal cardiac function. We will perform untargeted metabolomics profiling in a rat BC model of DIC to examine early plasma metabolite markers, visualize their spatial location in the heart tissue, and then identify changes in patterns of metabolite profiles in BC patients to test the hypothesis that DOX produces a ?metabotype? consisting of oxidized lipids and metabolites that can be used as early indicators of DIC in BC patients. Our Specific Aims are: 1) Determine the effects of DOX on cardiac function and metabolic profiles of the plasma and heart in the MATBIII rat model of BC; and 2) Determine the dynamic changes in plasma metabolomic profiles of BC patients that are associated with DIC. The proposed research will have a significant impact by identifying plasma metabolic markers of early anthracycline cardiotoxicity in plasma. Moreover, information on metabolic alterations in heart tissue will advance our biologic and mechanistic understanding of DIC through metabolic pathways that are perturbed in patients with BC.